Journal: Pflugers Archiv : European journal of physiology
Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes
doi: 10.1007/s00424-022-02767-8
Figure Lengend Snippet: A. Immunoblot detection of APOL1 in podocytes treated in the absence (-INFγ, rightmost lane) or presence of INFγ (Ctrl, leftmost lane). APOL1 is not induced by IFNγ in three clonal populations derived from Celprogen human primary podocytes (C1KO, C2KO, C3KO) in which the APOL1 gene was subjected to CRISPR-mediated inactivation. β-actin served as loading control. B. Preservation of differentiated phenotype in Celprogen podocytes. Upper panels: immunoblot of nephrin in lysates of HEK-293 cells transiently overexpressing nephrin (lane 1), in SV40LgT-immortalized human podocytes (lane 2), in Celprogen primary human podocytes (lane 3) and in podocyte outgrowths from human glomeruli. Lower panels: immunoblots of WT1 and podocin in HEK-293 cells transiently overexpressing WT1 or podocin (lane 1), in SV40LgT-immortalized human podocytes (lane 2) and in Celprogen primary human podocytes (lane 3). β-actin served as loading control for both upper and lower panels. C. Whole cell IV curve (linear ramp from +100 (at left) to −100 mV (at right)) recorded from a representative human primary podocyte incubated 18 h in the absence (lower trace) or presence of IFNγ (10 ng/mL, upper trace). D. Whole cell IV curve (linear ramp from +100 (at left) to −100 mV (at right), recorded first in the absence (red trace) and then after 2 min in the presence of anti-APOL1 aa 1–238 antibody (black trace +Ab), and finally after 3 min of antibody washout (black trace “washout”). Seal resistances at the end of each period were 9.7 GΩ (-Ab), 8.3 GΩ (+Ab) and 4.0 GΩ (washout). E. Capacitance-normalized whole cell currents measured at −60 mV in normal human primary podocytes incubated 24 h in the absence (n=11, left three datasets) or presence of 10 ng/ml IFNγ (n=19, right three datasets); *, p<0.016 vs. minus IFNγ. Each cell was recorded first in the absence and then in the subsequent presence of anti-APOL1 N-terminal domain antibody for 2 min (2 μg/ml, +Ab; n=6 −IFNγ and n=11 +IFNγ), followed by 3 min antibody washout (w/o, n=3 −IFNγ and n 5 +IFNγ). Among the 19 IFNγ-pretreated cells, open circles represent cells for which patch seals did not survive the antibody exposure, black circles connected by black lines depict cells for which patch seals did not survive antibody washout, and black circles connected by red lines show cells for which patch seals survived all three recording conditions. #, p= 0.135, vs. −IFNγ before antibody; ##, p=0.008 vs. +IFNγ before antibody; one-way ANOVA on ranks with Dunnett’s post-test.
Article Snippet: Rabbit polyclonal antibody to human WT1 was from Proteintech (12609–1-AP).
Techniques: Western Blot, Derivative Assay, CRISPR, Control, Preserving, Incubation
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